PROJECT SUMMARY Mosquito-transmitted alphaviruses cause outbreaks of incapacitating acute and chronic arthritis and life- threatening encephalitis. The arthritogenic alphaviruses include the re-emerging chikungunya (CHIKV), Mayaro, and Ross River viruses. Since 2004, CHIKV has infected millions of people and expanded into Europe, Asia, and Americas. As arthritis can endure for months to years after infection with an arthritogenic alphavirus, large epidemics have severe economic consequences. The encephalitic alphaviruses include Eastern, Venezuelan (VEEV), and Western equine encephalitis viruses that are endemic to the Americas and infection can lead to mortality or long-term neurological sequelae. There are currently no approved alphavirus- specific vaccines or antiviral agents. In collaboration with Southern Research, we used HTS to identify non- toxic small molecules that inhibit CHIKV and VEEV replication. Additionally, we identified chemically distinct compound classes that display broad inhibitory activity against alphaviruses, as well as other pathogenic human viruses (e.g., flaviviruses). We also mapped resistance mutants against two highly active compounds and identified the alphavirus nsP2 helicase domain and the nsP3 macrodomain as potential antiviral targets. In addition to this work, in collaboration with the Emory Institute for Drug Development, we identified novel nucleosides, which target RNA-dependent RNA polymerases, with potent antiviral activity against alphavirus infection. Thus, we have identified potent antiviral compounds against three distinct molecular targets essential for alphavirus replication. The goal of this highly interactive Project 2 of the Antiviral Drug Discovery and Development Center (AD3C) is to develop new therapeutic strategies that inhibit alphavirus replication, prevent selection for drug resistance, and reduce alphavirus disease severity. In Specific Aim 1, lead compounds will be optimized in collaboration with Core A and Core B by iterative medicinal chemistry, cell culture-based antiviral and cytotoxicity assays, and in vivo pharmacokinetic studies to improve their efficacy, selectivity, solubility, and bioavailability. In Specific Aim 2, we will work with the other AD3C Projects to define the breadth of antiviral activity and cell type specificity, molecular targets through resistance mapping and structural/computational analyses and identify synergy profiles for compounds with potent activity. In Specific Aim 3, optimized compounds will be evaluated for in vivo efficacy using well-established pre-clinical mouse models of alphavirus infection. Since therapies that target different aspects of the viral life cycle are likely to show improved efficacy and limit the development of drug resistance, combination therapies also will be tested. Last of all, since alphavirus-induced disease includes inflammatory immune pathology, we will evaluate the therapeutic potential of combining antiviral therapy with anti-inflammatory regimens. This work will promote the development of new antiviral therapeutic strategies that have the potential to improve human health.